Glycoproteins having lipid mobilizing properties and therapeutic applications thereof

ABSTRACT

A biologically active lipid mobilizing agent for use in therapy is disclosed which has the properties and characteristics of a Zn-α 2 -glycoprotein, or of a fragment thereof having an apparent molecular mass M r  greater than 6.0 kDa as determined by gel exclusion chromatography. Methods of isolation and purification from biological material are also disclosed together with uses of the material for making up pharmaceutical compositions, especially pharmaceutical compositions useful for treating mammals to achieve weight reduction or for controlling obesity. In addition, uses of the material for developing diagnostic agents and for identifying inhibitors of lipolytic activity for therapeutic purposes are disclosed.

The above patent application is the U.S. National Stage ofPCT/GB99/01509, filed Jun. 1, 1999, which claims priority from GreatBritain Patent Application No. 9811465.5, filed May 29, 1998, both ofwhich are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

This invention relates to the field of biochemistry and medicine and itis especially concerned with therapeutic applications of certainglycoproteins, including fragments thereof, which exhibit lipidmobilising properties in biological systems. In particular, in oneaspect the invention embraces the use of such glycoproteins andfragments thereof for therapeutic treatment of mammals to achieve aweight reduction or for controlling obesity. The invention also relatesto the isolation and purification of such glycoproteins from biologicalmaterial. The invention also relates to the use of such glycoproteinsfor developing diagnostic agents and inhibitors for therapeutic use.

BACKGROUND

For convenience, reference publications relating to or mentioned in thefollowing description are numerically labelled and listed in theappended bibliography.

The invention has its origins in research carried out in connection withcancer cachexia. Cancer cachexia is a common condition in many humancancer patients, especially patients with gastrointestinal or lungcancer, and is characterised by progressive weakness, dramatic weightloss and wasting resulting from loss of both adipose tissue and skeletalmuscle mass. Previous investigations have indicated that thecharacteristic loss of weight and body issues (fat and muscle) cannotusually be explained simply by a reduction of food and water intake, andthe effect has been attributed to production by the tumours of catabolicfactors that pass into the circulatory system. Both lipolytic andproteolytic activities are involved, and there have been numerousattempts to isolate and purify the substances that produce theseactivities, especially lipid mobilising factors responsible for thecatabolism of adipose tissue and reduction of carcass fat.

In GB2217330A, for example, the supposed isolation and purification wasdescribed of lipolytic factors derived from a cachexia-inducing murinetumour designated MAC16 and also from the urine of cachectic cancerpatients using chromatographic methods which included at least one stageof gel filtration exclusion chromatography, and results were obtainedthat suggested there were several related molecular species having anapparent molecular weight less than 5000 daltons that were responsiblefor the lipolytic effect. Severe problems were encountered, however, inattempting to purify the active molecular species to the extent requiredfor use in therapeutic applications and fully to characterise the activematerial in terms of its chemical constitution. More recently, in 1995,a paper by T. M. McDevitt et al., entitled “Purification andcharacterisation of a lipid-mobilising factor associated withcachexia-inducing tumours in mice and humans”, was published in CancerResearch 55, 1458-1463 (reference 1), wherein it was reported that amaterial having an apparent relative molecular mass M_(r) of 24 kDa hadbeen isolated from both the above-mentioned cachexia-inducing murinetumour MAC16 and from the urine of patients with cancer cachexia usingan isolation and purification procedure involving a combination of ionexchange, size exclusion and hydrophobic chromatography, and a beliefwas expressed that this material was a purified form of cancer cachexialipid mobilising factor. It was subsequently found, however, that this24 kDa material was in fact a proteoglycan which when purified tohomogeneity would produce a cachectic state in non-tumour bearing miceby inducing catabolism of skeletal muscle protein, as reported by P.Todorov et al. 1996, Nature, 379, 739-742 (reference 2). Thus, this 24kDa material was a proteolytic factor and it seems that any lipolyticactivity had to be attributed to contamination through co-purificationwith a separate and distinct lipolytic factor.

SUMMARY OF THE INVENTION

The present invention is based on the subsequent finding that a truelipolytic or lipid mobilising factor (LMF) produced by thecachexia-inducing murine tumour MAC16 and present also in urine ofcancer cachexia patients is in fact a glycoprotein which has an apparentrelative molecular weight of about 43 kDa as determined by itselectrophoretic mobility when subjected to 15% SDS-PAGE electrophoresisand which is the same as, or which is very similar to and hascharacteristics in common with, a glycoprotein known asZn-α₂-glycoprotein. Zn-α₂-glycoprotein has been known since it was foundin human blood plasma and first reported in a paper by Burgi and Schmidentitled “Preparation and properties of Zn-α₂-glycoprotein of normalhuman plasma” (1961) J. Biol. Chem. 236, 1066-1074 (reference 3).Although the properties and physiological function of this material havenot been fully determined, the material has been highly purified andcharacterised in terms of chemical and physical chemical properties.Moreover, the complete amino acid sequence has been reported in a paperentitled “Complete amino acid sequence of human plasmaZn-α₂-glycoprotein and its homology to histocompatibility antigens” byT. Araki et al. (1988) Proc. Natl. Acad. Sci. USA., 85, 679-683(reference 4) wherein the glycoprotein was shown as consisting of asingle polypeptide chain of 276 amino acid residues having threedistinct domain structures (A, B and C) and including two disulfidebonds together with N-linked glycans at three glycosylation sites. Thisamino acid sequence of the polypeptide component is set out in FIG. 1 ofthe accompanying drawings. Although some subsequent publications haveindicated that the composition of human Zn-α₂-glycoprotein can varysomewhat when isolated from different body fluids or tissues, allpreparations of this material have substantially the same immunologicalcharacteristics. As reported by H. Ueyama, et al. (1991) “Cloning andnucleotide sequence of a human Zn-α₂-glycoprotein cDNA and chromosomalassignment of its gene”, Biochem. Biophys. Res. Commun. 177, 696-703(reference 5), cDNA of Zn-α₂-glycoprotein has been isolated from humanliver and prostate gland libraries, and also the gene has been isolated,as reported by H. Ueyama et al. (1993) “Molecular cloning andchromosomal assignment of the gene for human Zn-α₂-glycoprotein”,Biochemistry 32, 12968-12976 (reference 6). H. Ueyama et al. have alsodescribed, in J. Biochem. (1994) 116, 677-681 (reference 7), studies onZn-α₂-glycoprotein cDNA's from rat and mouse liver which, together withthe glycoprotein expressed by the corresponding mRNA's, have beensequenced and compared with the human material. Although detaildifferences were found as would be expected from different species, ahigh degree of amino acid sequence homology was found with over 50%identity with the human counterpart (over 70% identity within domain Bof the glycoprotein). Again, common immunological properties between thehuman, rat and mouse Zn-α₂-glycoproteins have been observed.

The preparation of purified Zn-α₂-glycoprotein from fresh human plasmaby a method involving six steps of column chromatography separation hasbeen described by Ohkubo et al. in a paper entitled “Purification andcharacterisation of human plasma Zn-α₂-glycoprotein” (1988) Prep.Biochem., 18, 413-430 (reference 8), of which the content isincorporated herein by reference.

The 43 kDa glycoprotein lipolytic or lipid mobilising factor (LMF)isolated and purified in connection with the present invention has beenobtained substantially free of any proteolytic factor, both from thecachexia inducing murine tumour MAC16 and from urine of patients withcancer cachexia, using an improved isolation and purification procedure.This procedure has again involved a combination of ion exchange,exclusion and hydrophobic chromatographic separations but theselectivity of the separations differs from that of chromatographicseparations previously used when the 24 kDa cachectic factor wasisolated, yielding a product that when subjected to 15% SDS-PAGEelectrophoresis shows a single band of apparent relative molecularweight of about 43 kDa. As already indicated, the lipolytic activematerial or lipid mobilising factor (LMF) thus isolated, from both theMAC16 tumour and from cancer patients' urine, has been found to be aglycoprotein with characteristics in common with or the same as those ofZn-α₂-glycoprotein isolated from human plasma. Accordingly it has beenconcluded that this human and mouse LMF are both Zn-α₂-glycoproteins orare very close analogues thereof having a substantial degree of sequencehomology and substantially the same biological activity, especially inrelation to lipolytic activity with respect to adipocytes. They maytherefore be referred to as glycoproteins of the Zn-α₂-glycoproteintype.

In particular, it has been found that:

-   a) the human and mouse lipid mobilising factors which have been    isolated from the above-mentioned sources both co-migrated with    authentic human plasma Zn-α₂-glycoprotein on 15% SDS-PAGE and on 10%    non-denaturing gels;-   b) the human and mouse lipid mobilising factors isolated both    stained heavily for carbohydrates in the same way as authentic    Zn-α₂-glycoprotein;-   c) a polyclonal antibody against human plasma Zn-α₂-glycoprotein was    capable of detecting the lipid mobilising activity of the human    material and of neutralising this activity in vitro;-   d) authentic human plasma Zn-α₂-glycoprotein also shows in vitro    lipid mobilising activity and also stimulates adenylate cyclase    activity;-   e) the human and mouse lipid mobilising factor and the authentic    human Zn-α₂-glycoprotein each show the same chymotrypsin digestion    pattern producing similar fragments and loss of activity;-   f) the human lipid mobilising factor isolated is homologous with    authentic human plasma Zn-α₂-glycoprotein in amino acid sequence and    both have been shown to stimulate production of adenylate cyclase in    murine adipocycte plasma membranes in a GTP-dependent process with    maximum stimulation at 0.1 μMGTP.

The term authentic Zn-α₂-glycoprotein is used herein to denote purifiedZn-α₂-glycoprotein as prepared from fresh human plasma substantiallyaccording to the method described by Ohkubo et al. (reference 8). Itwill be appreciated that in some cases fragments of the isolated lipidmobilising factor or of authentic Zn-α₂-glycoprotein may be producedwithout loss of the lipolytic or lipid mobilising activity, and variousadditions, deletions or substitutions may be made which also will notsubstantially affect this activity. In that aspects of the presentinvention relate to therapeutic applications, it is however importantthat a high degree of purity should generally be achieved and, inparticular, the material should be substantially free of proteolyticactivity.

In one aspect, the present invention relates to the use in medicine of aglycoprotein lipid mobilising factor as herein defined or atherapeutically effective fragment derived therefrom for treatment ofconditions of overweight or obesity in mammals.

More particularly, the invention provides a biologically active lipidmobilising agent for use in therapy characterised in that it has theproperties and characteristics of a Zn-α₂-glycoprotein, or of a fragmentof a Zn-α₂-glycoprotein that has an apparent molecular mass M_(r), asdetermined by gel exclusion chromatography, greater than 6.0 kDa. Inpreferred embodiments this lipid mobilising agent can be defined asbeing a glycosylated polypeptide wherein the polypeptide moiety isselected from one of the following groups:

-   -   (a) a polypeptide having the amino acid sequence of a        Zn-α₂-glycoprotein;    -   (b) a polypeptide which in respect to (a) is deficient in one or        more amino acids;    -   (c) a polypeptide in which in respect to (a) one or more amino        acids are replaced by a different amino acid or acids;    -   (d) a polypeptide in which in respect to (a) there is a        plurality of additional amino acids which do not interfere with        the biological lipolytic activity or which may be readily        eliminated;    -   (e) a polypeptide which is an allelic derivative of a        polypeptide according to (a).

Also according to the invention, a biologically active lipid mobilisingagent for use in therapy consists essentially of a glycoprotein, or afragment of said glycoprotein that has an apparent relative molecularmass M_(r), as determined by gel exclusion chromatography, greater than6 kDa, said glycoprotein being characterised in that it has apolypeptide amino acid sequence that is homologous with the amino acidsequence (SEQ ID No: 1) of human plasma Zn-α₂-glycoprotein, or with avariant thereof which is modified by additions, deletions, orsubstitutions that do not substantially affect its lipid mobilisingactivity in biological systems.

In at least some embodiments of the invention the lipid mobilising agentmay be further characterised by an apparent relative molecular massM_(r) of about 43 kDa as determined by its electrophorectic mobilitywhen subjected to 15% SDS-PAGE electrophoresis.

Thus, also according to the invention, a purified biologically activelipid mobilising agent for use in therapy is characterised in that itconsists essentially of a glycosylated polypeptide comprising a singlemain component having an apparent relative molecular mass M_(r) of about43 kDa as determined by its electrophoretic mobility when subjected to15% SDS-PAGE electrophoresis and having homology in amino acid sequencewith the amino acid sequence (SEQ ID No: 1) of human plasmaZn-α₂-glycoprotein. This lipid mobilising agent may be furthercharacterised in some embodiments by the fact that it can be obtained bya process that includes sequential steps of subjecting biologicalmaterial to ion exchange chromatography, exclusion chromatography, andthen to hydrophobic interaction chromatography, wherein said biologicalmaterial is a body fluid of a cancer cachexia patient or an extract of aculture of a MAC16 tumour cell line deposited in the name of MichaelJohn Tisdale under the provisions of the Budapest Treaty in the EuropeanCollection Of Animal Cell Cultures (ECACC) ) at the Public HealthLaboratory Service Centre for Applied Microbiology and Research,Portondown, Salisbury, Wiltshire, United Kingdom, under an Accession No.89030816.

Also, in at least some embodiments, the lipid mobilising agent of thepresent invention may be further characterised by one or more of thefollowing features:

-   -   (a) when subjected to digestion with chymotrypsin it is        fragmented and its lipid mobilising properties are destroyed;    -   (b) it has the potential in vitro to stimulate adenylate cyclase        activity in a guanine triphosphate (GTP) dependent process upon        incubation with murine adipocyte plasma membranes;    -   (c) it has substantially the same immunological properties as        human Zn-α₂-glycoprotein;    -   (d) it is an active lipid mobilising fragment of the aforesaid        43 kDa glycoprotein or glycosylated polypeptide obtainable by        digesting the latter with trypsin;    -   (e) it is substantially free of proteolytic activity;    -   (f) the polypeptide chain of the polypeptide component has an        N-terminus blocked by a pyroglutamate residue;    -   (g) the lipid mobilising activity is destroyed by periodate        treatment.

The invention also provides pharmaceutical compositions for use intreating mammals, e.g. to reduce their weight or control obesity, saidcompositions containing as the active constituent an effectivetherapeutic amount of Zn-α₂-glycoprotein or glycoprotein lipidmobilising factor as herein defined, or a lipolytically active fragmentthereof, together with a pharmaceutically acceptable carrier, diluent ofexcipient.

The invention also includes the use of a lipid mobilising agent, asherein defined, for the manufacture of a medicament useful in humanmedicine for treating conditions of overweight or obesity.

Thus, the invention further provides a glycoprotein lipid mobilisingfactor having properties and characteristics of Zn-α₂-glycoprotein,especially human Zn-α₂-glycoprotein, for use in the production of amedicament effective in treating conditions of overweight or obesity.Such a medicament may also be useful for stimulating muscle developmentand increasing muscle mass.

The invention also provides a method of isolating and purifyinglipolytically active glycoprotein or lipid mobilising agent having theproperties and characteristics of a Zn-α₂-glycoprotein, i.e. aglycoprotein of the Zn-α₂-glycoprotein type, said method comprisingsubjecting an extract of a cachexia-inducing tumour or of a culture of acachexia-inducing tumour cell line, or a sample of urine or other bodyfluid from a mammal bearing a cachexia-inducing tumour, to a combinationof ion exchange, gel filtration or size exclusion chromatography, andhydrophobic interaction chromatography, yielding a single product ormolecular species having an apparent molecular weight or relativemolecular mass of 43 kDa, as determined by 15% SDS-PAGE electrophoresis,which is substantially free of proteolytic activity.

The invention also includes a method of treating a mammal to bring abouta weight reduction or reduction in obesity, said method comprisingadministering to the mammal a therapeutically effective dosage of alipid mobilising agent as herein defined. In general, this will beprovided by a glycoprotein identical to or homologous with a humanZn-α₂-glycoprotein, or an effective fragment thereof, substantially freeof any proteolytic activity.

The lipid mobilising glycoprotein or Zn-α₂-glycoprotein may beadministered as an injectable formulation incorporating a carrier in theform of a pharmaceutically acceptable injection vehicle.

The glycoprotein or fragment thereof used in these therapeuticapplications may further be produced by recombinant DNA techniques suchas are well known in the art, based possibly on the known cDNA sequencefor Zn-α₂-glycoprotein which has been published for example in reference7.

The invention also includes a method for detecting the presence of acachexia inducing tumour, and/or for monitoring changes in such atumour, e.g. during the course of antitumour therapy, said methodcomprising taking a sample of urine, blood serum or other body fluid,and testing to detect the presence of and/or to measure the amounttherein of the lipid mobilising agent herein defined or ofZn-α₂-glycoprotein. In carrying out this method, a monoclonal orpolyclonal antibody against Zn-α₂-glycoprotein or other biochemicalreagent may be used as a diagnostic detecting agent, as hereinafterdescribed.

The purified lipid mobilising factor or Zn-α₂-glycoprotein of thisinvention may also be used for producing antibodies, either monoclonalor polyclonal antibodies but preferably monoclonal antibodies, which canthen be used as diagnostic detecting agents as mentioned above, or whichcan be used in therapy as inhibitors or antagonists to the lipolyticagent(s) causing cachexia in cancer patients.

The antibodies referred to may be, for example, whole antibodies orfragments thereof Particular antibody fragments may include thoseobtained by proteolytic cleavage of whole antibodies, such as F(ab′)₂,Fab′ or Fab fragments; or fragments obtained by recombinant DNAtechniques, for example Fv fragments (as described in InternationalPatent Specification No. WO 89/02465. In a further aspect of theinvention, the use of one or more of such antibodies is envisaged forthe manufacture of a medical preparation or medicament for the treatmentof cachexia-associated cancer and/or tumours.

The antibody or antibody fragment may in general belong to anyimmunoglobulin class. Thus, for example, it may be an immunoglobulin M(IgM) antibody or, in particular, an immunoglobulin G (IgG) antibody.The antibody or fragment may be of animal, for example mammalian, originand may be for example of murine, rat or human origin. It may be anatural antibody or a fragment thereof, or, if desired, a recombinantantibody or antibody fragment, i.e. an antibody or antibody fragmentwhich has been produced using recombinant DNA techniques.

Particular recombinant antibodies or antibody fragments include, (1)those having an antigen binding site at least part of which is derivedfrom a different antibody, for example those in which the hypervariableor complementary determining regions of one antibody have been graftedinto the variable framework regions of a second, different antibody (asdescribed in European Patent Specification No. 239400); (2) recombinantantibodies or fragments wherein non-Fv sequences have been substitutedby non-Fv sequences from other, different, antibodies (as described inEuropean Patent Specifications Nos. 171496. 172494 and 194276); or (3)recombinant antibodies or fragments possessing substantially thestructure of a natural immunoglobulin but wherein the hinge region has adifferent number of cysteine residues from that found in the naturalimmunoglobulin, or wherein one or more cysteine residues in a surfacepocket of the recombinant antibody or fragment is in the place ofanother amino acid residue present in the natural immunoglobulin.(asdescribed in International Patent Specifications Nos. WO 89/01974 and WO89/01782 respectively).

As indicated, the antibody or antibody fragment may be polyclonal, butis preferably of monoclonal origin. It may be polyspecific, but ispreferably monospecific for the lipolytic material or Zn-α₂-glycoproteinof the invention.

Whole antibodies may be prepared using well-known immunologicaltechniques employing the purified active lipolytic material orZn-α₂-glycoprotein from any source as antigen. Thus, for example, anysuitable host may be injected with the lipolytic material and the serumcollected to yield the desired polyclonal antibody after appropriatepurification and/or concentration (for example, by affinitychromatography using immobilised lipolytic material as the affinitymedium). Alternatively, splenocytes or lymphocytes may be recovered fromthe injected host and immortalised using for example the method ofKohler et al., (1976), Eur. J. Immuno, 6, 511, (reference 9) theresulting cells being segregated to obtain a single genetic lineproducing monoclonal antibodies in accordance with conventionalpractice.

If in the above methods the lipolytic material is of a size that doesnot elicit a suitable immune response in the host, even though it may beantigenic and capable of binding to specific antibodies, it may bepreferable covalently to link the material to a large carrier moleculewhich is itself immunogenic, and to +use the resulting conjugatecompound as the antigen, again in accordance with conventional practice[see for example, D. M. Weir, in “Handbook of Experimental Immunology”,3, 2^(nd) ed. pp A2.10-A2.11. Blackwell Scientific Publications, Oxford,1973, (reference 10); and M. Z. Atassi and A. F. S. A. Habeeb, in“Immuno-chemistry of Proteins” (M. Z. Atassi, ed), 2, pp 177-264,Plenum, New York, 1977 (reference 11)].

Antibody fragments may be produced using conventional techniques, forexample by enzymatic digestion, e.g. with pepsin [Lanoyi and Nisonoff,(1983) J. Immunol. Meth., 56, 235, (reference 12)]. Where it is desiredto produce recombinant antibodies according to the invention these maybe produced using for example the general methods described in theabove-mentioned patent specifications.

The invention also extends to diagnostic kits for carrying out thediagnostic methods referred to, such kits comprising a receptacle forreceiving the sample of body fluid, a biochemical reagent for detectingsaid lipid mobilising agent or Zn-α₂-glycoprotein, and instructions foruse of the kit.

The lipid mobilising agent of the present invention may also be used forscreening and identifying and/or for carrying out investigations ofpossible lipolytic activity inhibiting agents having potential asanti-cachectic or antitumour therapeutic agents. This screening may becarried out by adding samples of possible antagonists to, or inhibitorsof, the activity of said lipid mobilising agent to preparations of saidlipid mobilising agent, followed by incubation in vitro with apreparation of adipocytes and assaying to determine the level oflipolytic activity relative to that of a control sample.

MORE DETAILED DESCRIPTION

Examples hereinafter presented illustrate in more detail at least someaspects of the invention and its development. There first follows,however, an outline or summary of some of the materials, methods andtechniques which have generally been used in the development of theinvention and in the illustrative examples unless subsequently statedotherwise.

Animals:

Pure strain NEW and ob/ob mice were bred from existing in-housecolonies; mate BKW mice (40-50 g) were purchased from Banting andKingman, Hull, United Kingdom. These animals were transplanted withfragments of the MAC16 tumour into the flanks, by means of a trocar asdescribed by S. A. Beck et al. (1987) “Production of lipolytic andproteolytic factors by a murine tumour-producing cachexia in the host”Cancer Res. 47, 5919-5923 (reference 14). The solid tumours were excisedfrom the mice when the weight loss reached 25%.

Subjects:

Urine was collected from patients having unresectable pancreatic cancerwith established weight loss ranging between 1.3 and 10 kg/month. Thesepatients were not receiving therapy at the time of urine collection.Samples of urine were stored frozen at −20° C. in the absence ofpreservatives prior to the purificiation.

Chromatography Apparatus and Materials:

Sephadex™ Mono Q HR 5/5 anionic exchange resin, Superose™ 12H 10/30 gelexclusion and Resource™ Iso hydrophobic chromatography columns werepurchased from Pharmacia Biotech, St. Albans, United Kingdom. AnAquapore™ AX-300 DEAE-cellulose column was supplied by AppliedBiosystems, California. Rainbow™ protein molecular weight markers, ECLWestern blotting system and Hyperfilm™-ECL autoradiography film werefrom Nycomed Amersham Plc, United Kingdom.

Other Materials:

Other materials included a DIG glycan detection kit from BoehringerMannheim GmbH, Germany, protein A peroxidase conjugate from Sigma,Dorset, United Kingdom, nitrocellulose membranes from Hoefer ScientificInstruments, California and Amicon filters (YM10) from Amicon Ltd.,Stonehouse, Gloucestershire, United Kingdom. Also used were“Mini-Message Maker” and spot-on kits purchased from Rand D Systems,Abingdon, United Kingdom and Superscript™ TH11 RT reverse transcriptasefrom Gibco BRL, Paisley, Scotland. Oligonucleotides were synthesized byOswell, Southampton, United Kingdom.

DEAE Cellulose Column Chromatograghy:

In a typical example of using this technique, homogenate containingactive lipid mobilising factor (LMF) would be centrifuged and thesupernatant would be fractionated by anion exchange chromatography usinga DEAE-cellulose column and eluting under a salt gradient. TheDEAE-cellulose column would first be equilibrated with buffer solutionat the required pH before applying a sample of the material to befractionated. Thereafter, material would be eluted from the column usinga linear salt gradient, e.g. 0 to 0.2M NaCl, in the same buffer. Theeffluent from the column would be collected in small volume fractions,e.g. 5 ml fractions, and the lipolytic activity of each fraction wouldbe measured by the lipolytic assay technique referred to below.

Use of a DEAE cellulose column with elution under a salt gradient is aprocedure at least potentially useful as a preliminary separation stage,but it can be especially useful for obtaining further fractionationafter a stage of gel filtration exclusion chromatography and prior to afinal or later purification stage of hydrophobic interactionchromatography. As hereinafter described, in a subsequent stage orstages the latter may be carried out employing selected hydrophobicchromatography columns such as Resource™ Iso columns in conjunction withhigh performance liquid chromatography (HPLC) methods.

Serum Metabolite Determinations

Non-esterified fatty acids (NEFA) were determined using a Wako-ASC-ACODkit (Wako Chemical GmbH, Neuss, Germany). Triglycerides were determinedusing a Triglyceride kit (Sigma Chemical Co., Poole, United Kingdom) and3-hydroxybutyrate by a quantitative enzymatic determination kit (Sigma).Glucose was measured using a glucose analyser (Beckman, Irvine, Calif.)and glycerol was determined enzymatically using the method of Wieland asdescribed in “Methods of Enzymatic Analysis” (Ed. Bergmeyer, H. U.) Vol.3, pp1404-1409, published by Academic Press, London (1974) (reference13).

Lipolytic Assay

Single cell suspensions of white adipocytes were prepared from finelyminced epididymal fat pads of male BKW mice using collagenase digestion,substantially as described by S. A. Beck et al. (see above-mentionedreference 14). Samples to be assayed were incubated with 10⁵-2×10⁵adipocytes (determined by means of a haemocytometer) for 2 h at 37° C.in 1 ml of Krebs-Ringer bicarbonate buffer, pH 7.2. The concentration ofglycerol released was determined enzymatically by the method of Wielandas referred to above (see also GB2217330A). Control samples containingadipocytes alone were analyzed to determine the spontaneous glycerolrelease. Lipid mobilizing activity was expressed as μmol glycerolreleased/10⁵ adipocytes/2 h.

Isolation of Human Omental Adipocytes

Human omental adipose tissue was removed under general anaesthesia andtransported immediately to the laboratory. Fragments of tissue (roughlyequivalent in size to a pair of murine epididymal fat pads) weredigested to produce a single cell suspension of adipocytes by incubationat 37° C. for 30 min in a 1 ml aliquot of Krebs-Ringer bicarbonatebuffer supplemented with 4% bovine serum albumin, 1 g/l glucose and 1.5mg/ml collagenase, using for this purpose a shaking water bath.

Isolation of Mouse Adipocyte Plasma Membranes

In a typical procedure white adipocytes were isolated from mouseepididymal fat pads as referred to above except that the cells werewashed in 250 mM sucrose, 2 mM ethyleneglycolbis(β-aminoethylether)-N,N,N′,N′ (EGTA), 10 mM Tris-HCl (pH 7.4).Adipocytes were resuspended in 20 ml of the above buffer and homogenisedby aspirating through a Swinny filter at least 10 times. The cellhomogenate was then centrifuged at 300 g for 5 min, the fat cake removedfrom the surface and the remaining pellet and infranatant transferred toclean tubes. These were centrifuged at 30,000 g for 1 h at 4° C. and themembrane pellet formed was resuspended in the sucrose buffer (200 to 400μl). Plasma membranes were separated from other organelle membranes on aself-forming gradient of Percoll™ colloidial silica particles. Theconstituents were 250 mM sucrose, 2 mM EGTA, 10 mM Tris-HCl, pH 7.4;Percoll™; and 2M sucrose, 8 mM EGTA, 80 nM Tris-HCl, pH 7.4, mixed in aratio of 32:7:1 together with the membrane suspension (in a total volumeof 8 ml). This mixture was centrifuged at 10,000 g for 30 min at 4° C.The gradient was fractionated into 0.75 ml portions and each portion wasassayed for the presence of succinate dehydrogenase, NADH-cytochrome creductase, lactate dehydrogenase and 5′-nucleotidase to locate theplasma membrane fraction. The membrane fractions were resuspended in 150mM NaCl, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4 and centrifuged at 10,000 gat 4° C. for 2 min. The process was repeated twice. The washed plasmamembranes were then diluted in 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2mM EGTA and 4 μM phenylmethylsulfonyl fluoride (PMSF) at 1-2 mg/ml, snapfrozen in liquid nitrogen and stored at −70° C. until use.

Adenylate Cyclase Assay

An adenylate cyclase assay used was based on that developed by Salomonet al. (reference 16). Briefly, water (negative control), isoprenaline(positive control) or LMP was added to an assay mix (final volume 100μl) containing 25 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, GTP (guaninetriphosphate), 8 mM creatine phosphate, 16 units/ml creatinephosphokinase, 1 mM 3-isobutyl-1-methylxanthine and 1 mM [α-³²P] ATP(sp. act. 20 mCi/mmole). Preincubation was at 30° C. for 5 min and thereaction was initiated by the addition of plasma membrane (typically 50μg protein). After 10 min at 30° C. the reaction was terminated by theaddition of 100 μl of a solution containing 2% sodium dodecylsulphate,40 mM ATP and 1.4 mM cyclic AMP. In order to determine recovery ofcyclic AMP [8-³H] adenosine 3′,5′-cyclic phosphate (1 μCi in 50 μl ofwater) was added to each tube. Background binding was determined byrunning samples without [α-³²P] ATP and sample controls were set upwithout plasma membranes.

Samples containing labelled nucleotides were diluted to 1 ml with waterand loaded onto Dowex™ 50W8-400 ion-exchange columns primed with 10 mMof water. After washing twice with 1 ml of water the cyclic AMP waseluted with 3 ml of water into polypropylene tubes containing 200 μl of1.5M imidazole, pH 7.2. The samples were then applied to Alumina WN-3columns (previously washed with 8 ml of 0.1M imidazole, pH 7.5) and theeluate collected directly into scintillation vials containing thescintillation fluid supplied under the Trade Mark Optiphase HiSafe 3. Afurther 1 ml of 0.1M imidazole was added to the columns and the eluatewas combined with the run through. The radioactivity was determinedusing a Tri-carb™ 2000A scintillation analyser.

Zn-α₂-glycoprotein

Samples of Zn-α₂-glycoprotein were used in identifying the lipidmobilizing factor isolated. The Zn-α₂-glycoprotein used was purifiedapproximately 670-fold from fresh human plasma using a combination ofDEAE-Sephadex A-50, DEAE-Sephacel, Zn-chelate Sepharose 6B,Phenyl-Sepharose, Sephacryl S-300 and HA-Ultrogel column chromatographysubstantially as described by Ohkubo et al. “Purification andcharacterisation of human plasma Zn-α₂-glycoprotein” (1988) Prep.Biochem 18, 413-430 (reference 8), of which the content is incorporatedherein by reference.

Gel Electrophoresis

Gels were prepared according to the method of Laemmli (reference 15) andgenerally consisted of a 5% stacking gel and a 15% SDS-PAGE resolvinggel (denaturing or reducing conditions) or a 10% SDS-PAGE resolving gel(non-denaturing or non-reducing conditions). Samples were loaded at 1-5μg/lane. Bands were visualised by staining either with Coomassiebrilliant blue R-250 or by silver. Samples were prepared for reducingconditions by heating for 5 min at 100° C. in 0.0625M Tris-HCl, pH 6.8,10% glycerol, 1% SDS, 0.01% bromophenol blue and 5% 2-mercaptoethanol.

For immunoblotting, the gels were transferred to nitrocellulosemembranes which had been blocked with 5% Marvel in 0.15% Tween 20 in PBSat 4° C. overnight. The nitrocellulose membranes were washed once for 15min and twice for 5 min in 0.5% Tween 20 in phosphate buffered saline(PBS) at room temperature. Immunodetection was carried out usingpolyclonal antiserum for Zn-α₂-glycoprotein (10 μg/ml) prepared asdescribed by Ohkubo et al. (see reference 8 mentioned above) in 1.5%Marvel, 0.15% Tween 20 in PBS for 1 hour at room temperature. Afterbeing washed three times as above the filters were incubated for 1 hourwith protein A peroxidase conjugate at a 1:500-fold dilution followed byone 15 min wash and four 5 min washes with 0.5% Tween 20 in PBS. The ECLdetection system was used, and the blots were suspended in equal volumesof detection reagents 1 and 2 using 0.125 ml/cm² for 1 min at roomtemperature and then wrapped in Saran Wrap™. The blots were exposed toautoradiography film (Hyperfilm™ ECL) for 30 seconds to 10 min dependingon the amount of target protein.

BRIEF DESCRIPTION OF THE DRAWINGS

In connection with the description of the invention and illustrativeexamples detailed below reference should be made to the accompanyingdrawings in which:

FIG. 1 is a diagram of the complete amino acid sequence (SEQ ID No: 1)of the human plasma Zn-α₂-glycoprotein, as published by T. Araki et al.(1988) “Complete amino acid sequence of human plasma Zn-α₂-glycoproteinand its homology to histocompatibility antigens” (reference 4);

FIG. 2 is a diagram of the lipolytic activity distribution pattern andprotein content of fractions obtained in a stage of anion exchangechromatography, using an Aquapore™ AX-300 DEAE column, applied to theactive lipolytic fractions obtained from a preliminary stage of gelfiltration chromatographic separation on a Q-Sepharose column ashereinafter described in Example 2;

FIG. 3 is a diagram of the lipolytic activity distribution pattern andprotein content of fractions obtained by a further stage of HPLChydrophobic interaction chromatography on a Resource™ Iso hydrophobiccolumn of those fractions from the Aquapore™ AX-300 DEAE fractionationstage illustrated in FIG. 2 that contained the major activity peak;

FIG. 4 shows the electrophoresis patterns produced by human and mouseLMF isolated and purified as in Examples 1 and 2, and also the patternproduced by human plasma Zn-α₂-glycoprotein following 15% SDS-PAGE;

FIG. 5 is a diagram similar to FIG. 4 but shows the banding patternobtained for human plasma Zn-α₂-glycoprotein (lane 2) and for human LMF(lane 3) as prepared from Example 2;

FIG. 6 shows further banding patterns obtained with SDS-PAGE used fordetecting carbohydrate as is also hereinafter described;

FIG. 7 shows a Western blot banding pattern produced by human plasmaZn-α₂-glycoprotein (lane 1) and by human LMF (lane 2) after 15% SDS-PAGEusing a polyclonal antibody to Zn-α₂-glycoprotein;

FIG. 8 is a further electrophoresis banding pattern obtained followingexperiments made to determine the effect of α-chymotrypsin on humanplasma Zn-α₂-glycoprotein and on the isolated and purified human LMF;

FIG. 9 is a bar chart diagram comparing the stimulation of lipolysis infreshly isolated murine epididymal adipocytes by human LMF (A) and byhuman Zn-α₂-glycoprotein (B), the results being expressed as a mean±SEMValues for glycerol release from fat cells alone have been subtractedfrom the values given, and the data is representative of three separateexperiments. Differences from controls were determined by Student'st-test and are indicated as *p≦0.05, **p≦0.01 and ***p≦0.005.

FIG. 10 is a diagram showing change in body weight of ex-breeder maleNMRI mice (30-40 g) produced by intravenous (iv) administration of LMF(8 μg) isolated from human urine as described in Example 2 (o) and ofcontrol mice administered PBS by iv injection (x).

FIG. 11 is a diagram similar to FIG. 10 showing change in body weight ofob/ob mice produced by iv administration of LMF (35 μg) (o) isolatedfrom human urine as described in Example 2 and of control mice (x)administered PBS by iv injection. LMF was injected at times 0, 16, 24,40, 48, 64, 72, 90, 96, 113, 120, 137 and 144 h; PBS was injected at thesame time points. The animals were killed 160 h after the firstinjection. Results are expressed as mean±SEM for 5 animals per group.

FIG. 12 shows graphs illustrating the effect of trypsin digestion fordifferent time periods (2 hrs, 4 hrs and 8 hrs) on the biologicalactivity of the 43 kDa LMF.

EXAMPLE 1 Isolation and Purification of Lipid Mobilizing Factor fromMurine Adenocarcinoma MAC16.

The procedure followed in this example is summarised in Table 1 at theend of the present description and involved the initial purification ofthe lipid mobilising factor (LMF) from the MAC16 tumour using apreliminary batch extraction on DEAE-cellulose and/or possibly proteinprecipitation by ammonium sulphate, followed by anion exchangechromatography on a Sepharose™ Mono Q HR 5/5 anion exchange column andsize exclusion on Superose 12.

More particularly, solid tumours were excised from mice with weight lossand homogenized in 10 mM Tris-HCl (pH 8.0) containing 0.5 mMphenylmethylsulfonyl fluoride (PMSF), 0.5 mM EGTA, and 1 mM DTT at aconcentration of 5 ml/g of tumour. Debris was removed from thehomogenate by low-speed centrifugation (4000 rpm for 15 min in abench-top centrifuge). When using ammonium sulfate precipitation,ammonium sulphate solution (38% w/v) was slowly added at this stage tothe supernatant with stirring at 4° C. and the precipitate was removedby centrifugation (4500 rpm for 20 min). The supernatant was thenconcentrated using an Amicon filtration cell containing a membranefilter with a molecular weight cut-off of M_(r) 10,000 against originalhomogenisation buffer.

Batch extraction on DEAE-cellulose at this stage this was convenientlycarried out substantially as described by T. M. McDevitt et al.“Purification and characterization of a lipid-mobilizing factorassociated with cachexia inducing tumours in mice and humans” CancerRes., (1995) 55, 1458-1463 (reference 1).

The next step was anion exchange chromatography using Q-Sepharose. Thecolumn used was the Mono Q HR 5/5 anion exchange column which has aprotein capacity of 20-50 mg protein. The column was equilibrated priorto use with homogenising buffer, and the sample was loaded aftercentrifugation for 10 min at 1300 rpm as a 500-μl injection. After aninitial wash, active material was eluted under a 0-0.2 M NaCl gradient.The presence of the active fractions was determined using themeasurement of glycerol release from murine adipocytes in accordancewith the lipolytic bioassay previously referred to. The active fractionswere concentrated using an Amicon filtration cell and dissolved in 0.5ml of 50 mM phosphate (pH 8.0) containing 0.3 M NaCl, 0.5 mM PMSF, 0.5mM EGTA, and 1 mM DTT prior to FPLC Superose™ (or Superdex™)chromatography. The column used in carrying out this specific examplewas the Superose 12 prepacked 10/30 gel exclusion column, which wasequilibrated with the above buffer for 2 hours at 0.25 ml/min, afterwhich the sample was loaded as a 200-μl injection. Thirty 1.0 mlfractions were collected, and the lipid-mobilising activity was detectedby the aforesaid lipolytic bioassay.

Up to this point the procedure has followed closely that described byMcDevitt et al. in previously mentioned reference 9, but whereas thelatter then continued with a final step of HPLC using a C₈ hydrophobiccolumn and an acetonitrile/TFA gradient, in the present case the activefractions from the Superose column were further fractionated using anAquapore™ AX-300 DEAE-cellulose column coupled to an HPLC system andeluting under a gradient from 0 to 0.3M NaCl before carrying out thefinal HPLC hydrophobic chromatography stage using the hydrophobic columnmarketed under the Trade Mark “Resource Iso”. This modification led tothe isolation of a much more stable bioactive product different from theproducts previously isolated.

In this last-mentioned stage of HPLC using the Aquapore™ AX-300DEAE-cellulose column, typically the flow rate was 0.2 ml min⁻¹ with asolvent system composed of component A (10 mM Na-phosphate pH 5.3) andcomponent B (10 mM Na-phosphate pH 5.3+0.3 M NaCl). All solvents weredegassed prior to use. In one particular separation the gradient waschanged according to the following protocol: 10 min 0%B, 40 minutes100%B, 50 minutes 100%B, and 60 minutes 0%B. Absorbance (A₂₁₄) wasmonitored at 214 nm to determine protein content Each of the elutedpeaks was collected as a separate fraction. The salt in these fractionsfrom the DEAE-cellulose column was removed by ultrafiltration through aMicrocon™ micro-concentrator containing a membrane filter having amolecular weight cut-off of M_(r) 10,000 (Amicon) against deionizedwater containing 0.5 mM PMSF, 0.5 mM EGTA, 1 mM DTT. Again, thelipid-mobilising activity was detected by the lipolytic bioassay, andactive fractions were concentrated using a Microcon™ microconcentratoragainst 50 mM phosphate buffer (pH 7.0) containing 1.5M ammonium sulfateprior to HPLC hydrophobic chromatography.

In the HPLC final step of this purification procedure using thehydrophobic column Resource™Iso, 1 ml (Pharmacia Biotech), the flow ratewas 1 m/min⁻¹ with a solvent system C (50 mM phosphate pH 7.0+1.5 Mammonium sulfate) and D (50 mM phosphate pH 7.0). All solvents weredegassed prior to use. A typical gradient protocol was 5 minutes 0%D, 20minutes 100%D, 30 minutes 100%D, and 35 minutes 0%D. Again, absorbance(A₂₁₄) was monitored at 214 nm to determine protein content, and each ofthe eluted peaks was collected as a separate fraction. After removingthe salt by ultrafiltration (through a Microcon™ microconcentratoragainst deionized water containing 0.5 mM PMSF, 0.5 mM EGTA, and 1 mMDTT) the lipid-mobilising activity was detected by the lipolyticbioassay as before.

EXAMPLE 2 Isolation and Purification of a Lipid-mobilising Factor fromUrine of Cachectic Patients

Urine from a cancer patient with weight loss was fractionated accordingto a scheme similar to that used for the MAC16 tumour, although fewersteps were required to get a pure product (see summary in Table 2 at theend of the present description) because of the lower protein content ofurine. In the first stage the urine was subjected to precipitation with80% (NH₄)₂SO₄ and the precipitate was dialysed against 10 mM Tris-HCl(pH 8.0) containing 0.5 mM PMSF, 0.5 mM EGTA and 10 mM DTT using anAmicon filtration cell containing a membrane filter having a molecularweight cut-off of M_(r) 10,000. The urine concentrate was thenfractionated by anion exchange chromatography using Q-Sepharose,followed by HPLC using an Aquapore™ AX-300 (30×21 mm) DEAE-cellulosecolumn (flow rate of 0.2 ml min⁻¹ with 10 mM phosphate buffer at pH 5.3)under a linear 0-0.4M NaCl gradient which was run for 30 minutes. Theprotein content and the bioactivity of the fractions were determinedrespectively by measuring the absorbance A₂₁₄ and by measuring therelease of glycerol from epididymal adipocytes using the standardlipolytic assay as previously described. The results of typicalfractionation at this DEAE-cellulose fractionation stage are illustratedin the diagram of FIG. 2.

There then followed a final stage of hydrophobic interactionchromatography using the hydrophobic column Resource™Iso (6.4×30 mm) tofractionate the active material obtained from the DEAE-cellulose column,substantially as described in connection with Example 1. In this finalstage, typically the starting buffer was 50 mM phosphate, pH 7.0,containing 1.5M (NH₄)₂SO₄ and the column was run under a linear gradientof the elution buffer (50 mM phosphate, pH 7.0, with a flow rate of 1 mlmin⁻¹). The diagram of FIG. 3 illustrates the results in one example ofthis final stage of hydrophobic chromatography.

Upon repeating the procedure of Example 2 on a range of cancer patientsand normal subjects it was found that although cancer patients withweight loss generally show the presence of this LMF in the urine it wasabsent from the urine of cancer patients without weight loss and fromnormal subjects, as demonstrated for example in Table 3 at the end ofthe present description.

Properties and Identity of the Lipid-Mobilising Factor (LMF) as Isolatedin Examples 1 and 2 A. Molecular Weight

When subjected to 15% SDS-PAGE, both the human and mouse LMF, isolatedand purified as described, showed a single protein band of an apparentrelative molecular mass of M_(r) 43 kDa. This is illustrated in FIG. 4in which lane 1 shows molecular weight markers, lanes 3 and 4 show thebanding pattern obtained with the human LMF and lane 5 shows the bandingpattern obtained with the mouse LMF.

When electrophoresed on 10% non-denaturing PAGE the purified human andmouse LMF both showed an apparent molecular weight of 84 kDa, thebanding pattern obtained using the human LMF being shown in lane 3 ofFIG. 5 wherein lane 1 again shows molecular weight markers.

B. Structure and Comparison with Zn-α₂-glycoprotein

Sequence analysis of both the human and mouse LMF material revealed thatit comprised a polypeptide chain having an N-terminus blocked by apyroglutamate residue. Treatment with HCl or pyroglutamateaminopeptidase to remove this residue, or cleavage with chymotrypsin,produced peptides that showed homology with human plasmaZn-α₂-glycoprotein in residues 2-6, 55-79 and 146-167 (see Araki et al.,reference 4). Purified human and mouse LMF also comigrated withZn-α₂-glycoprotein when electrophoresed on 15% SDS-PAGE as illustratedin FIG. 4 in which lane 2 shows the banding pattern obtained usingauthentic human Zn-α₂-glycoprotein (prepared as described in reference8). The purified human LMF and human Zn-α₂-glycoprotein also had thesame molecular weight (84,000) on 10% non-denaturing PAGE (see lanes 3and 2 respectively in FIG. 5). Using SDS-PAGE for detection ofcarbohydrate, both human and mouse materials stained heavily as did alsoauthentic human Zn-α₂-glycoprotein. This is illustrated in FIG. 6 inwhich lane 1 shows the effect of human plasma Zn-α₂-glycoprotein; lane 2the effect of human LMF; lanes 3 and 4 the results with mouse LMF; lane5 the result with transferrin (positive control); lane 6 the result withcreatinase (negative control). The gel was stained for carbohydrateusing the DIG glycan detection kit according to the manufacturer'sinstructions.

It was also found that a polyclonal antibody raised against authentichuman plasma Zn-α₂-glycoprotein was capable of detection of human LMF onimmunoblots, as shown in FIG. 7, and of neutralisation of in vitro lipidmobilizing activity of the human, but not the mouse material. The latteris indicated in Table 4, and an explanation of this observation may liein the fact that mouse Zn-α₂-glycoprotein has been shown to exhibit only58.6% identity in amino acid sequence with the human counterpart (seereference 7).

FIG. 8 shows the effect of α-chymotrypsin on authentic human plasmaZn-α₂-glycoprotein and LMP. Lane 1 shows molecular weight markers; lane2 shows human plasma Zn-α₂-glycoprotein; lane 3 shows human LMF; lane 4shows the result of Zn-α₂-glycoprotein+α-chymotrypsin; lane 5 representshuman LMF+α-chymotrypsin; and lane 6 is α-chymotrypsin alone (control).Proteins were electrophoresed on 15% SDS-PAGE and stained with Coomassiebrilliant blue. Both human plasma Zn-α₂-glycoprotein and the isolatedand purified LMF showed the same chymotryptic cleavage fragments andchymotrypsin destroyed the in vitro biological activity of the LMF.Neither human plasma Zn-α₂-glycoprotein nor the human LMF contained theM_(r) 24 kDa proteolysis inducing factor (PIF) previously reported toco-purify with the LMF.

The expression of Zn-α₂-glycoprotein in various murine tumours and liverhas also been quantitated by competitive PCR. Liver, being known toexpress Zn-α₂-glycoprotein, was used as a control for the tumours. Ofthe MAC tumours evaluated only the cachexia-inducing MAC16 was found toexpress Zn-α₂-glycoprotein.

C. Biological Activity

C.1 In vitro

The human LMF material isolated from the urine of cancer patients withweight loss as in Example 2 was tested at different doses for itslipolysis stimulating effect on freshly isolated murine epididymaladipocytes by measuring glycerol release using the lipolytic assaypreviously described. The test was also repeated using authentic humanplasma Zn-α₂-glycoprotein and it was found that both the authenticZn-α₂-glycoprotein and the human LMF material stimulated glycerolrelease with a comparable dose-response profile. This is illustrated inFIG. 9 where diagram A shows the results for the human LMF at differentconcentrations and diagram B shows the results for the authentic humanplasma Zn-α₂-glycoprotein.

Induction of lipolysis in adipocytes is thought to be mediated by anelevation of the intracellular mediator cyclic AMP, and in further testsit was found that incubation of murine adipocyte plasma membranes withthe human LMF caused a stimulation of adenylate cyclase activity in aGTP-dependent process, with maximal stimulation occurring at 0.1 μM GTP.Also, this activation of adenylate cyclase was found to be saturablewith concentrations of LMF >5 μg/assay. Using human plasmaZn-α₂-glycoprotein it was found that this also stimulated murineadipocyte plasma membrane adenylate cyclase in a GTP-dependent mannerwith maximal stimulation also at 0.1 μM GTP. Again, this activation ofadenylate cyclase by the Zn-α₂-glycoprotein was found to be saturablewith concentrations >5 μg/assay.

This data showing the similar effects and comparable dose-responseprofiles for LMF and Zn-α₂-glycoprotein, together with the ability ofpolyclonal antisera to Zn-α₂-glycoprotein to neutralise in vitrolipolysis by human LMF, the homology in amino acid sequence, andmatching electrophoretic mobility, all provide strong evidence that theisolated and purified LMF is indeed Zn-α₂-glycoprotein. Althoughprevious reports have shown that Zn-α₂-glycoprotein is an adhesiveprotein closely related to antigens of the major histocompatibilitycomplex in amino acid sequence and domain structure, there have been noprevious reports of a capacity of Zn-α₂-glycoprotein to inducelipolysis. Moreover, it has not previously been reported as beingpresent in human urine. At present the mechanism by which a large acidicprotein such as Zn-α₂-glycoprotein can stimulate adenylate cyclase isnot known and is quite surprising since other known substances having asimilar role are small and basic polypeptides.

C.2 In vivo

In order to determine if the purified LMF isolated from human urine asin Example 2 was capable of fat depletion in vivo a sample of this LMFmaterial (8 μg) was injected into male ex breeder NMRI mice over a 72 hperiod. The LMF was injected at times 0, 17, 24, 41, 48, 62 and 72 hoursand control mice were similarly injected with phosphate-buffered saline(PBS) at the same time points. The animals were killed at 89 h and thebody composition and serum metabolite levels were determined. As shownin FIG. 10, there was a progressive decrease in body weight of theanimals receiving LMF which was significantly lower than PBS treatedcontrols within 41 h of treatment. Changes in the body composition andserum metabolic levels are summarised in Table 5 and it will be seenthat total body weight decreased by 3.6 g during the overall 89 h periodof the experiment without change in food and water intake. Bodycomposition analysis showed a large reduction (42%) in the body fatcontent of mice receiving LMF, with a tendency to increase the non-fatmass although this did not reach a particularly significant level. Inthis connection some evidence has in fact been found indicating that theLMF may actually stimulate protein synthesis and thus increase musclemass. Despite the fat mobilisation there were significant reductions inthe serum concentrations of non-esterified fatty acids (NEFA), glyceroland glucose in mice receiving LMF.

As shown in FIG. 11 and by the data in Table 6, intravenousadministration to obese ob/ob mice of LMF (35 μg) isolated from humanurine produced a similar result. There was a decrease in total bodyweight which became significant within 24 h of the first injection andremained below that of the control group over the 160 h of theexperiment. Body composition analysis showed weight loss to arise from adecrease in carcass fat (26.03±0.70 g in controls and 21.09±0.99 g inLMF treated animals) without an alteration in the water content ornon-fat mass (see Table 6). Serum levels of glycerol and3-hydroxybutyrate were significantly increased, while blood glucoselevels were decreased and there was no effect on either triglyceride orNEFA levels.

D. Fragmentation

It was also established that the active 43 kDa glycoprotein could bedigested with trypsin to give a fragment of apparent molecular weight orrelative molecular mass of 7 kDa (as determined by gel filtrationexclusion chromatography using a Sephadex™ 50 column) which stillretains the biological activity of functioning as a lipid mobilisingagent. This is illustrated by the results of a typical experimentdepicted in FIG. 12 in which samples of the isolated human LMF wereincubated with trypsin at 37° C. for different time periods and thenanalysed by Sephadex—50 Gel Exclusion Chromatography and lipolyticassay. The results clearly indicate the presence of active fragmentswithin the 2.5 to 2.7 fractions, the M W of these fragments, as deducedfrom a calibration curve, being 6 kDa, 7 kDa and 8 kDa respectively asshown on the figure. Positive and negative controls were performed,these being as follows: −ve=0.027, +ve=0.252.

Therapeutic Use

Overall the results referred to in Section C.2 above in connection withthe in vivo experiments confirmed an increased metabolism of fat andshowed that in these model systems the isolated and purified human LMFproduces a decrease in carcass weight specifically by depletion ofadipose tissue. It is this particular ability of the human LMF, which isthe same as or a close analogue of Zn-α₂-glycoprotein, to reduce adiposetissue without affecting muscle mass that most clearly demonstrates thepotential for use of this material for the treatment of obesity inhumans. As has been mentioned earlier, there is also some evidenceindicating that this LMF material can actually stimulate proteinsynthesis and may therefore be useful for stimulating muscle developmentPotentially, the material is also especially useful for treating humanswith increased susceptibility to maturity onset diabetes such as canoccur in cases of obesity.

For this therapeutic use, particularly for the controlled treatment ofobesity in humans, either for medical reasons or cosmetic reasons, atherapeutically useful and non-toxic quantity of the essentially pureactive substance, either a lipid mobilising factor isolated and purifiedsubstantially as herein described or the equivalent purified orsynthetic Zn-α₂-glycoprotein, or material constituting a lipolyticallyactive fragment derived from the latter, can be made up as apharmaceutical formulation for administration in any suitable manner.Such formulations may be presented in unit dosage form and may comprisea pharmaceutical composition, prepared by any of the methods well knownin the art of pharmacy, in which a preparation of the active lipolyticsubstance is combined in intimate association or admixture with anyother suitable ingredient providing a compatible pharmaceuticallyacceptable carrier, diluent or excipient. The formulations include thosesuitable for oral, rectal, topical and parenteral (includingsubcutaneous, intramuscular and intravenous) administration. Forparenteral administration the formulations may comprise sterile liquidpreparations of a predetermined amount of the active lipolytic substancecontained in ampoules ready for use.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compoundin the form of a powder or granules; or as a suspension of the activecompound in an aqueous liquid or non-aqueous liquid such as a syrup, anelixir, an emulsion of a draught. The active compound may also bepresented as a bolus, electuary or paste.

The amount of the active compound which is required in order to beeffective for treating obesity in mammals will of course vary and isultimately at the discretion of the medical or veterinary practitionertreating the mammal in each particular case. The factors to beconsidered by such practitioner, e.g. a physician, include the route ofadministration, type of pharmaceutical formulation; the mammal's bodyweight, surface area, age and general condition.

Diagnostic Applications

For diagnostic purposes, to detect the presence of a tumour in a humanpatient or to monitor the progress of a tumour under treatment,basically it is only necessary simply to take a sample of body fluidsuch as urine in which Zn-α₂-glycoprotein is not normally present inhealthy individuals, and then to test this for the presence of theglycoprotein lipid mobilising agent or lipolytic factor (or equivalentZn-α₂-glycoprotein) herein identified.

In practice, any convenient method may be used for detecting and/ormeasuring this active lipid mobilising agent or lipolytic factor in thesamples, and the apparatus and materials required may advantageously bepackaged and supplied, together with appropriate practical instructions,in the form of self-contained diagnostic kits ready for immediate use.Particularly preferred diagnostic agents for detecting and/or measuringthe active lipid mobilising or lipolytic factor in a convenient andreliable manner are biochemical reagents, such as monoclonal orpolyclonal antibodies for example, capable of specifically recognisingand binding to human Zn-α₂-glycoprotein and then being identifiable by,for example, a visual change or a special screening using an associatedlabelled marker molecule, or by any other suitable technique known inthe art.

Monoclonal Antibodies

The production of monoclonal antibodies to the Zn-α₂-glycoprotein orZn-α₂-glycoprotein like lipolytic factor of this invention can beachieved by the use of established conventional techniques commonly usedin the art Such monoclonal antibodies, once prepared, may be immobilizedon suitable solid supports (in a column for example) and then used foraffinity purification to prepare in a convenient manner any furtherquantities that may be required for testing of the purified activelipolytic factor from tumour extracts or body fluids.

It is envisaged, however, that another important use of such monoclonalantibodies, apart from their use as a diagnostic agent, will be atherapeutic application based on their properties as inhibitors orantagonists to the active lipolytic factor in human cancer patients anda consequent therapeutic value as agents for treating and suppressingthe symptoms of cachexia and/or for preventing or reducing tumourgrowth. Thus, by virtue of this property, they can provide therapeuticagents and, more specifically, they can be used to make or manufacture amedical preparation or medicament for the therapeutic treatment ofcancer-associated cachexia and/or malignant tumours in mammals.

Screening Applications

Apart from monoclonal antibodies as referred to above, it is likely thatany agent which is antagonistic to, or an inhibitor of, the activity ofthis lipid mobilising or lipolytic factor of the present invention couldhave at least potential human therapeutic value. Hence, preparations ofthe purified, or at least partially purified, lipolytic factor (LMF)herein identified can be particularly useful, in accordance with afurther aspect of the invention, for use in providing a convenient invitro method of screening substances to find potential anti-cachecticand/or antitumour agents for therapeutic use. A typical example of thisapplication using freshly prepared adipocytes from mouse epididymaladipose tissue is outlined below:

The experiments are set up as follows:

-   -   100 μl purified LMF preparation+1 ml fat cells    -   Compound to be screened+1 ml fat cells    -   100 μl LMF preparation and compound+1 ml fat cells

Each compound is tested at increasing concentrations and all samples areprepared and processed in duplicate.

The samples are gassed for 2 min with 95% O₂, 5% CO₂ mixture, mixed andincubated for 2 hour at 37° C. After 2 hour, 0.5 ml from each sample isthen assayed for glycerol content as hereinbefore described.

Compounds which appear to show some significant degree of inhibition canthen be candidates for further evaluation.

In general, the inhibitory effect observed in such in vitro experimentscan be expected to occur also in vivo, and it is anticipated that byusing this screening method further antagonists or inhibitors will befound that will have useful therapeutic applications for the treatmentof cancer-associated cachexia and/or as antitumour agents.

MAC16 Cell Line and Purification

Although it is quite feasible for preparations containing useful amountsof the purified or partially purified active lipid mobilising orlipolytic factor to be produced as herein described from extracts oftumours, such as the MAC16 adenocarcinoma, grown in vivo, or from urineof cancer cachexia patients, or by synthetic methods, a more convenientand preferred alternative source may be provided by extracts of tumourtissue cell cultures, especially cultures of the MAC16 cell linepreviously referred to.

The cells of this cell line can be conveniently grown in RPMI 1640 mediacontaining 10% foetal calf serum under an atmosphere of 10% CO₂ in air.When assayed in the adipocyte glycerol release assay method it has beenfound that such culture grown cells may release a greater amount ofglycerol than do corresponding amounts of the tumour in vivo.

As will be seen, the invention presents a number of different aspectsand it should be understood that it embraces within its scope all noveland inventive features and aspects herein disclosed, either explicitlyor implicitly and either singly or in combination with one another.Also, many detail modifications are possible and, in particular, thescope of the invention is not to be construed as being limited by theillustrative example(s) or by the terms and expressions used hereinmerely in a descriptive or explanatory sense. It is also pointed outthat insofar as the terms “lipid mobilising factor (LMF)”, “lipidmobilising agent” and “lipolytic factor” are used in the presentspecification, these terms are generally to be regarded as beingsynonymous and have the same meaning.

TABLE 1 Purification of Lipid-Mobilizing Factor from MAC 16 tumor TotalSpecific activity activity Puri- Total Recov- (μmol/ Re- (μmol/10⁵ fica-Purification protein ery 10⁵ adi- covery adipocytes/ tion stage (mg) (%)pocytes) (%) mg protein) fold Tumor 500 100 27 0.054 homogenate Batch102 20.4 1.21 100 0.0119 1 extraction on DEAE- cellulose Q-Sepharose 1.50.3 1.17 97 0.78 65 Superdex 0.61 0.1 1.12 93 1.84 154 HPLC 0.12 0.021.02 84 8.5 714 DEAE- cellulose HPLC 0.02 0.004 1 83 50 4201Resource-iso

TABLE 2 Purification of Lipid-Mobilizing Factor from Cancer PatientUrine Total Specific activity activity Puri- Total Recov- (μmol/ Re-(μmol/10⁵ fica- Purification protein ery 10⁵ adi- covery adipocytes/tion stage (mg) (%) pocytes) (%) mg protein) fold 80% 210 100 78.4 0.37(NH₄)₂SO₄ precipitation Q-Sepharose 0.2 0.1 1.19 100 5.95 1 HPLC 0.0360.017 1.15 97 31.9 5.4 DEAE- cellulose HPLC 0.007 0.003 1.15 97 164.327.8 Resource-Iso

TABLE 3 Relationship between weight loss and appearance of LMF in urineWeight loss Patient number Diagnosis (kg/month) LMF 1. Pancreatic cancer1.6 + 2. Chorangio carcinoma 4.2 + 3. Gastric cancer 3.0 + 4. Gastriccancer 2.2 − 5. Pancreatic cancer 0 − 6. Pancreatic cancer 4.6 + 7.Pancreatic cancer 1.5 + 8. Ovarian cancer 4.3 + 9. Rectal cancer 0.7 +10. Periampullary cancer 0.3 + (recurrence) 11. Colorectal cancer 0.5 −12. Hepatoma 1.4 + 13. Pancreatic cancer 4.0 + 14. Periampullary cancer0.8 − 15. Pancreatic cancer 1.3 + 16. Normal 0 −

TABLE 4 Effect of a polyclonal antibody to human Zn α₂-glycoprotein onhuman and mouse lipid mobilizing activity. μmole glycerol/10⁵ p (fromAddition adipocytes/2h factor alone) Human LMF 0.0062 ± 0.0002 HumanLMF + pAb 0.0013 ± 0.0012 0.03 Mouse LMF 0.0977 ± 0.02  Mouse LMF + pAb0.1082 ± 0.015 NS LMF (5 μg human or 10 μg mouse in PBS) were incubatedovernight with agitation at 4° C. with a polyclonal antibody (pAb) tohuman plasma Zn-α₂-glycoprotein (10 μg in PBS) and the lipid mobilizingactivity was determined as described in methods. Results are expressedas mean ± SEM for three determinations and the experiment was repeatedthree times. Differences from values in the absence of the pAb weredetermined by Student's t-test.

TABLE 5 The effect of LMF isolated from human urine on body weight, bodycomposition, food and water intake and serum metabolite levels inex-breeder male NMRI mice Parameter Control Treated P Final body weight(g) 35.5 ± 2.0 31.6 ± 2.2 0.01 Water (g) 22.0 ± 0.9 18.3 ± 0.8 NS NonFat (g)  7.7 ± 0.8  9.6 ± 0.9 NS Fat (g)  5.9 ± 0.6  3.4 ± 0.4 0.05 Foodintake (g/day)  8.0 ± 0.6  8.0 ± 0.2 NS Water intake (ml/day)  4.5 ± 0.8 4.4 ± 0.4 NS NEFA (mEq/l)  1.63 ± 0.09  0.95 ± 0.03  0.003 Glycerol(mM)  8.86 ± 0.51  6.73 ± 0.45 0.05 Triglyceride (mg/l)  0.323 ± 0.036 0.201 ± 0.027 NS Glucose (mg/100 ml) 223 ± 9    186 ± 0.08 0.02Material was administered to mice according to the schedule in FIG. 10.Values represent the mean ± SEM for 5 mice per group. Differences fromcontrol values were determined by Student's t-test.

TABLE 6 The effect of human LMF on body weight body composition, foodand water intake and serum metabolite levels in ob/ob mice 160 h afterthe first injection. Parameter Control Treated P Initial body weight (g)66.7 ± 4.2 67.9 ± 2.9 NS Final body weight (g) 73.1 ± 4.3 69.5 ± 4.30.01 Water (%) 50.3 ± 0.5 53.7 ± 1.1 NS Non Fat (%) 15.5 ± 0.9 17.4 ±1.0 NS Fat (%) 34.6 ± 0.6 30.6 ± 0.7 0.05 NEFA (mEq/l)  1.47 ± 0.12 1.45 ± 0.45 NS Glycerol (mM)  2.51 ± 0.28  5.31 ± 0.45 0.02Triglyceride (mg/l)  0.40 ± 0.05  0.49 ± 0.04 NS Glucose (mg/100 ml) 317± 11 260 ± 12 0.02 3-Hydroxybutyrate (mM)  0.30 ± 0.02  0.44 ± 0.01 0.001 Oxygen uptake  0.18 ± 0.06  0.55 ± 0.07  0.009 (μl/mg BAT/h)Material was administered to mice according to the schedule in FIG. 11.Values represent the mean ± SEM for 5 mice per group. Differences fromcontrol values were determined by Student's t-test.

References

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1. A method of treating a mammal to bring about a weigh reduction orreduction in obesity, said method comprising administering to the mammalin need of such treatment a therapeutically effective dosage of a lipidmobilizing agent which is a Zn-α₂-glycoprotein, the polypeptide moietythereof having the sequence as shown in SEQ ID NO:1.
 2. A method oftreating a mammal to bring about a weight reduction or reduction inobesity, said method comprising administering to the mammal in need ofsuch treatment a therapeutically effective dosage of a lipid mobilizingagent having an apparent molecular mass of greater than about 6 kDa, asdetermined by gel exclusion chromatography, and which is obtained bydigesting Zn-α₂-glycoprotein, the polypeptide moiety of which has thesequence shown in SEQ ID NO:1, with the enzyme trypsin.